1) I designed a new micro-method for complement fixation
test by means of a capillary pipette. 2) By this method, the complement-fixing antibodies in an individual mouse could be tested without taking its life. 3} The complement fixation titers in mice immunized with Japanese B encephalitis had a considerable individuality. 4) An adjuvant containing anhydrous lanoline and paraffin-oil,
when mixed with Japanese B encephalitis vaccine, was effective to potent complement-fixing antibody productions in mice to this antigen.</p

Hioka, T.

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 8, Issue 1 1952 Article 2JANUARY 1952Studies on Complement Fixation Test in aMouse Immuni-zed with Japanese BEncephalitis Virus.T. Hioka∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Studies on Complement Fixation Test in aMouse Immuni-zed with Japanese BEncephalitis Virus.∗T. HiokaAbstract1) I designed a new micro-method for complement fixation test by means of a capillarypipette. 2) By this method, the complement-fixing antibodies in an individual mouse could betested without taking its life. 3} The complement fixation titers in mice immunized with JapaneseB encephalitis had a considerable individuality. 4) An adjuvant containing anhydrous lanoline andparaffin-oil, when mixed with Japanese B encephalitis vaccine, was effective to potent complement-fixing antibody productions in mice to this antigen.∗Copyright c©OKAYAMA UNIVERSITY MEDICAL SCHOOLFrom the Department of Hygiene(Chief: Prof. M. Ogata).Studies on Complement Fixation Te3t in a MouseImmunized with Japanese 8 Encephalitis Virus.ByT. Hioka.Received for publication on Aug. 31, 1951.Japanese encephalitis virus was first inoculated into the mon-key's cerebrum by M. Hayashi l ), then was made able to be trans-mitted to the mouse similar to St. Louis encephalitis. Since then,mice have become universally used for serological tests in Japa~eseencephalitis.Complement fixation test in Japanese encephalitis virus is per-formed with antigens prepared from a mouse's infected brain, andis applied to diagnose such patients or differentiate this virus fromanother encephalitis viruses2, 3). In the latter, mice are used as 8:source 01 antisera. Because, by immunizing mice with the mouse' sinfected brain. formation ot antibodies aHined to brain tissues fromnormal mouse could be avoided.And also mice are used in order to examine the relationshipbetween the comp·lement fixing and neutralizing antibody produc-tion and the resistence following vaccination in experimental ence-phalitis infections4).In such cases mice were bleed from their heart, and the serawere gathered from several of them, because the serum from amouse was so little. But when considering individuality in mice, itis desirable to test the complement-fixing antibodies of everyindividual. For this purpose, I designed a micro-method for com-plemennt fixation test by means of a glass capillary pipette. Andby this method, I investigated what influence Freund's adjuvanthad on complement-fixing antibody productions in each mouse.immunized with Japanese B encephalitis virus'».Materials and Methods.I. Virus strain: - Nakayama strain of Japanese B encephali.:.1Hioka: Studies on Complement Fixation Test in a Mouse Immuni-zed withProduced by The Berkeley Electronic Press, 195278 T. Hioka:tis virus was used, which was shared through the kindness of ProfAndo ot Japanese National Institute of Health.2. Vaccine: - prepared trom mice'sbrains infected withJapanese B encephalitis. These were emulsified to 1/10 with0.85 %NaCl solution, and inactivided hy adding formalin in a pro-portion of 0.2%.3. Vaccine-adjuvant mixture -: - was prepared by mixing 1part of vaccine to 1 part of sterile anhydrous lanoline and 2 parts ofsterile paraHin-oil.4. Complement fixation test: - was carried out by the fol-lowing new micro-method.Antigen, complement solution and sensitized sheep red cellssU$pension were prepared by the method described by Ando"» ,without essential modification.0.15 - 0.2 cc of blood was obtained by amputating the mouse'stail. This blood was put into a glass micro-test tube with a interiordiameter of 0.5 cm. Bleeding from the tail was stopped by scorch..ing H On fire. The test tube was plugged with cotton, and kept in8 ice..box over night. About 0.05 cc of serum was obtained aftercentrifugation, and was diluted 4 times with saline solution Thismay conveniently be done by means of a small capillary pipette,fitted with a rubber teat pinched ~ilh Hoffman's cock, and markednear the point corresponding to the desired volume. The serumwas taken up by this pipette and 'allowed to run slightly up thetube so as to admit an air bubble, to serve as an index, then thepipette was dipped into saline solution, and a similar volume wasadmitted three times. The serum and saline solution were thenblown out into the micro-test tube, and mixed by drawing it in andout of the pipette. The test tube was plugged with cotton, andheated at 5rc for 30 minutes in a water-bath. A part of thisserum was again diluted successively 2 times with saline solutionby means of a capillary pipette marked about 0.05 cc, and withconcaved slide glasses. The diluted serum must be used rapidlyfor the following test to avoid evaporation.Complement fixation test was carried out by means of capillarypipettes like Figure I, which were bended in a obtuse angle at itswaist, and marked at about 0.03 cc. The previously stated dilutedserum, two units of complement and antigen were sucked up oneafter another into this pipette by the same method as used indiluting the serum. The tip of the pipette must be cleaned with2Acta Medica Okayama, Vol. 8 [1952], Iss. 1, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol8/iss1/2Metho~New micro-methodStudies on Complement Fixation Teat in a Mouse Immunized etc. 79ganze every time a solutionis taken in. The capillary pipette wasthen laid on the rack, and after the rubber teat was taken off, it waswell shaken to mix the three components. The air-bubbles couldbe -blown out by a- blast into a capillary pipette by means ofanother capillary pipette. TheFigure t. pipette was kept in a ice box;between 2 -_8°C for 18 - 20 hours.And then sensitized sheep redcells suspension wa~ 'taken up'twice to the mark ~nd mixed.A(ter the- pipette was left in anincubator at 30°C· for half anhour, the results' were read.Since_ results obtained withundiluted antisera, with seradiluted J : 2 are questionable, thedilutions used in my test wereover 1 : 4. Only 2, 3 or 4 plusfixations were considered signi-ficant.Res1,1lts.1. Examination of the micro-method for complementfixation test.the new micro-complement fixation method, using egg-albu-min and its antibody made in the rabbit, was tested and c~mpa~~dwith the ordinary method described by Andos>. The' results: wereas in Table I. Both methods will always give the same resultsVo-hen performed carefully.Table I. Complement fixation test of Anti-egg-albuminrabbit serum, with homologous antigen.Antiserum dilutions . Antigen I Serumcont. CO:1t.1-1-:-8---'-1-1-:--16-1-1-:-3-2-;1-1-:-6-4-1:-1-:-1-28-,-1-,-1-:4-11 : 4 .-O-r-d-in-a-r-y-m-e-t-h-od------'--4---4-----4- ---2-- --0---'\--0------04 4 4 2 0 10 0Dilution of antigen i3 1: 100. 4 = no hemolysis, 0 = complete hemolysis.,3Hioka: Studies on Complement Fixation Test in a Mouse Immuni-zed withProduced by The Berkeley Electronic Press, 195280 T. Hioka:11. The complement fixation tests in mice,which r~ceived repeated injections of JapaneseB encephalitis vaccine.10 mice were injected intraperitoneally with 0.2 cc of injectedmice brain vaccine every five day for ten times. 10 days after thelast injection, bloods were taken from their tails, and tested accord·ing to the method described beforehand. The results are as inTable H. Complement fixation titers of mice showed a differencebetween 1: 8 to J : 128 inspite of giving them a similar immuniz,a"tion dosage. No reactions were seen wi th normal brain antigen.Table 11. Complement fixation test in mice immunizedwith Japanese encephalitis virus.Antiserum dilutionsNo. Weight1: 41 1 : 811 : 1611 : 3211 : 64 \ 1: 128N S Titer1 20g 4 4 4 4 4 3 0 0 1: 1282 20.5 g 4 4' 4 4 4 2 0 0 1: 1283 18g 4 4 4 4 3 1 0 0 1: 644 17.5 g 4 4 4 4 3 1 0 0 1: 645 16.5 g 4 4 4 3 1 0 0 0 1: 326 22 g 4 4 4 3 0 0 0 0 1: 327 23 g 4 4 4 2 0 0 0 0 '1: 328 20g 4 4 3 I 1 0 0 0 0 1: 329 199 4 4 1I0 0 0 0 0 1:810 21 g 4 3 0I0 0 0 0 0 1: 8·N: Normal mouse brain cont., S: Serum cont.HI. The progrees of complement·fixing antibodyproduction in the mouse, when injected withJapanese B encephalitis vaccine incorporatedwith adjuvant.10 mice weighing between 15 g to 20 g were divided into twoequal groups, I and H. Group I received a single subcutaneousinjection of 1cc vaccine-adjuvant mixture (containing 0.25 cc ofvaccine). Group H was injected J cc of vaccine diluted 2 timeswith saline solution as a control. Mice were bleed every weekafter injection, and the complement fixation titers were measured.The results are as in Figure H. The individuality of the mouse i$4Acta Medica Okayama, Vol. 8 [1952], Iss. 1, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol8/iss1/2Studies on Complement Fixation Test in a Mouse Immunized etc. ;81seen in this case, too. But in general, the antibody titers in group 1reached higher levels, and were more sustained than those in groupU. where the antibodies disappeared in a month after injection.Figure 11. Complement fixation test in mice immunizedwith Japanese encephalitis VirUS.I .11 with adiuvant- - -0- - • - <> -. without adiuvant3121144):"::':7::-:--~z. - - - - - - - - - - - - - e- - •A, \, \, \, \, \" ,, ,, \, I ",P"",\ \\ \, \\ ,\ \IniectionI: 8I: 256I: 4t : 64I: 128.~ 1: 32:0~i:"~ ': 16"'ClEISDayeDiscussion.To study the complement fixation test in a mouse, it IS neces-sary to perform a test with very little serum. If a mouse could bebleed about 11100 of their body weight without evil effect to life;only about 0.15 cc blood could be taken from a mouse weighing15 g, and from this blood about 0.05 cc of serum could be obtained.And by the usual complement fixation method, this serum must behighly diluted for test. But by the above mentioned new micro-5Hioka: Studies on Complement Fixation Test in a Mouse Immuni-zed withProduced by The Berkeley Electronic Press, 1952T.Hioka:.method, the complement fixation test could be carried out withonly 4 times dilution of the serum. I designed this method by ahint taken from the technics used in Wright's opsonin test"'), andbended the capillary pipette at its waist so that the reaction couldbe performed in the pipette. This method could be applied tohuman beings, when it is difficult to obtain large quantity of bloodfrom the vein, and only a small amount of blood is obtainable fromthe ear-lobe.By using this method, I tested the so-called hyperimmune serumtaken from a mouse, injected with Japanese B encephalitis virus,and found that these immune sera had a considerable individualityin their complement fixati'on titers.By this method, I also succeeded in investigating the progressof immunity in a mouse injected with a mixture of Japanese B en-cephalitis vaccine and adjuvant. In 1942 Freund and McDermott;,)demonstrated that an adiuvant containing mineral oil, emulsifyingagents and killed acid-fast organismus, when mixed with horseserum, was especially effective in increasing the immune responseof rabbits to this antigen. The antibody response to a variety ofantigen has been potentiated in this way8). And on Japanese en-cephalitis, Warren and Hough9 ) carried out the protection testagainst infection in mice injected with a mixture of Fa/ba, mineraloil and vaccine made from infected mice brain, and reported thatin this case the minimal immunizing dosis could be decreased to1/3 as compared with the control which had no adjuvant. Accord-ingly, I selected anhydrous lanoline and paraffin-oil as adjuvant,and found that this adjuvant was effective in mice to potent com-plement-fixing antibody productions against Japanese encephalitisvirus.Summary.1) I designed a new -micro-method for complement fixationtest by means of a capillary pipette.2) By this method, the complement-fixing antibodies in anindividual mouse could be tested without taking its life.3} The complement fixation titers in mice immunized withJapanese B encephalitis had a considerable individuality.4) An adjuvant containing anhydrous lanoline and paraffin-oil,'when mixed with Japanese B encephalitis vaccine, was effective6Acta Medica Okayama, Vol. 8 [1952], Iss. 1, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol8/iss1/2Studies on Complement Fixation Test in a Mouse Immunized etc. 83to potent complement-fixing antibody productions ln~ mIce to thisantigen.In conclusion. I wish to express my sincere thanks to Prof. M. Ogata forhis kind advice and suggestions during this investigation.References.1 M. Hayashi. Proc. of Imp. Acad.• X, No. i. 1934; Folia Psychiatrica etNeur~logica japonica, Bd. 1. Heft~. Dec; 1935. - 2 Ashida. japan. J. Exp. Med.20. 489. 1950. - 3 Hughes and Perlowagora. J. Immun. 65. 155. 1950. - 4 Casals.j. Exp. Med. 78.447. 1943. - 5 Freund and McDermott. Proc. Soc. Exp. Bioi. &Med. 49, 548. 1942. - 6 Ando, Eisei Kensa Shishin II (edited by Welfare Mini-stry of japan. 1950). - 7 Wright. Technik von Gummisaugkappe und Glas-kapiIIare. (published by Gustav Fischer. 1914). - 8 7: Ogata. Igaku no Ayumi10. 349. 1950. - 9 Warren and Hough. Proc. Soc. Exp. BioI. & Med. 61. 109.1946.7Hioka: Studies on Complement Fixation Test in a Mouse Immuni-zed withProduced by The Berkeley Electronic Press, 1952

Studies on Complement Fixation Test in a Mouse Immunized with Japanese B Encephalitis Virus.

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Studies on Complement Fixation Test in a Mouse Immuni­zed with Japanese B Encephalitis Virus.

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Studies on Complement Fixation Test in a Mouse Immunized with Japanese B Encephalitis Virus.